Amplification of human MDM2 gene in human tumors

ABSTRACT

A human gene has been discovered which is genetically altered in human tumor cells. The genetic alteration is gene amplification and leads to a corresponding increase in gene products. Detecting that the gene, designated hMDM2, has become amplified or detecting increased expression of gene products is diagnostic of tumorigenesis. Human MDM2 protein binds to human p53 and allows the cell to escape from p53-regulated growth.

This application is a divisional application of Ser. No. 08/044,619,filed Apr. 7, 1993, now U.S. Pat. No. 5,420,263, which is acontinuation-in-part of Ser. No. 07/903,103, filed Jun. 23, 1992, nowU.S. Pat. No. 5,411,860, which is a continuation-in-part of Ser. No.07/867,840, filed Apr. 7, 1992, now abandoned.

FIELD OF THE INVENTION

The invention relates to the area of cancer diagnostics andtherapeutics. More particularly, the invention relates to the detectionof a gene which is amplified in certain human tumors.

BACKGROUND OF THE INVENTION

According to the Knudson model for tumorigenesis (Cancer Research, 1985,vol. 45, p. 1482), there are tumor suppressor genes in all normal cellswhich, when they become non-functional due to mutation, cause neoplasticdevelopment. Evidence for this model has been found in cases ofretinoblastoma and colorectal tumors. The implicated suppressor genes inthese tumors, RB and p53 respectively, were found to be deleted oraltered in many of the tumors studied.

The p53 gene product, therefore, appears to be a member of a group ofproteins which regulate normal cellular proliferation and suppression ofcellular transformation. Mutations in the p53 gene have been linked totumorigenesis, suggesting that alterations in p53 protein function areinvolved in cellular transformation. The inactivation of the p53 genehas been implicated in the genesis or progression of a wide variety ofcarcinomas (Nigro et al., 1989, Nature 342:705-708), including humancolorectal carcinoma (Baker et al., 1989, Science 244:217-221), humanlung cancer (Takahashi et al., 1989, Science 246:491-494; Iggo et al.,1990, Lancet 335:675-679), chronic myelogenous leukemia (Kelman et al,1989, Proc. Natl. Acad. Sci. USA 86:6783-6787) and osteogenic sarcomas(Masuda et al., 1987, Proc. Natl. Acad. Sci. USA 84:7716-7719).

While there exists an enormous body of evidence linking p53 genemutations to human tumorigenesis (Hollstein et al., 1991, Science253:49-53) little is known about cellular regulators and mediators ofp53 function.

Hinds et al. (Cell Growth & Differentiation, 1:571-580, 1990), foundthat p53 cDNA clones, containing a point mutation at amino acid residue143, 175, 273 or 281, cooperated with the activated ras oncogene totransform primary rat embryo fibroblasts in culture. These mutant p53genes are representative of the majority of mutations found in humancancer. Hollstein et al., 1991, Science 253:49-53. The transformedfibroblasts were found to produce elevated levels of human p53 proteinhaving extended half-lives (1.5 to 7 hours) as compared to the normal(wild-type) p53 protein (20 to 30 minutes).

Mutant p53 proteins with mutations at residue 143 or 175 form anoligomeric protein complex with the cellular heat shock protein hsc70.While residue 273 or 281 mutants do not detectably bind hsc70, and arepoorer at producing transformed foci than the 175 mutant, complexformation between mutant p53 and hsc70 is not required for p53-mediatedtransformation. Complex formation does, however, appear to facilitatethis function. All cell lines transformed with the mutant p53 genes aretumorigenic in a thymic (nude) mice. In contrast, the wild-type humanp53 gene does not possess transforming activity in cooperation with ras.Tuck and Crawford, 1989, Oncogene Res. 4:81-96.

Hinds et al., supra also expressed human p53 protein in transformed ratcells. When the expressed human p53 was immunoprecipitated with two p53specific antibodies directed against distinct epitopes of p53, anunidentified M_(r) 90,000 protein was coimmunoprecipitated. Thissuggested that the rat M_(r) 90,000 protein is in a complex with thehuman p53 protein in the transformed rat cell line.

As mentioned above, levels of p53 protein are often higher intransformed cells than normal cells. This is due to mutations whichincrease its metabolic stability (Oven et al., 1981, Mol. Cell. Biol.1:101-110; Reich et al. (1983), Mol. Cell. Biol. 3:2143-2150). Thestabilization of p53 has been associated with complex formation betweenp53 and viral or cellular proteins. (Linzer and Levine, 1979, Cell 17:43-52; Crawford et al., 1981, Proc. Natl. Acad. Sci. USA 78 :41-45;Dippold et al., 1981, Proc. Natl. Acad. Sci. USA 78:1695-1699; Lane andCrawford, 1979, Nature (Lond.) 278:261-263; Hinds et al., 1987, MolCell. Biol. 7 :2863-2869; Finlay et al., 1988, Mol. Cell. Biol.8:531-539; Sarnow et al., 1982, Cell. 28:387-394; Gronostajski et al.,1984, Mol. Cell. Biol. 4 :442-448; Pinhasi-Kimhi et al. 1986, Nature(Lond.) 320 182-185; Ruscetti and Scolnick, 1983, J. Virol. 46:1022-1026; Pinhasi and Oren, 1984, Mol. Cell. Biol. 4 :2180-2186; andSturzebecher et al, 1987, Oncogene 1 :201-211). For example, p53 proteinhas been observed to form oligomeric protein complexes with the SV40large T antigen, the adenovirus type 5 E1 B-M_(r) 55,000 Protein, andthe human papilloma virus type 16 or 18 E6 product. Linzer and Levine,1979, Cell 17 :43-52; Lane and Crawford, 1979, Nature, 278 :261-263;Sarnow et al., 1982, Cell 28 :387-394; Werness et al., 1990, Science,248 :76-79. Similarly, complexes have been observed of p105^(RB) (theproduct of the retinoblastoma susceptibility gene) with T antigen(DeCaprio et al., 1988, Cell 54:275-283), the adenovirus EIA protein(Whyte et al., 1988, Nature 334 :124-129) and the E7 protein of humanpapilloma virus 16 or 18 (Munger et al., 1989, EMBO J. 8:4099-4105). Ithas been suggested that interactions between these viral proteins andp105^(RB) inactivate a growth-suppressive function of p105^(RB),mimicking deletions and mutations commonly found in the RB gene in tumorcells. In a similar fashion, oligomeric protein complex formationbetween these viral proteins and p53 may eliminate or alter the functionof p53. Finlay et al., 1989, Cell 57:1083-1093.

Fakharzadeh et al. (EMBO J. 10:1565-1569, 1991) analyzed amplified DNAsequences present in a tumorigenic mouse cell line (i.e., 3T3DM, aspontaneously transformed derivative of mouse Balb/c cells). Studieswere conducted to determine whether any of the amplified genes inducedtumorigenicity following introduction of the amplified genes into anontransformed recipient cell (e.g., mouse NIH3T3 or Rat2 cells). Theresulting cell lines were tested for tumorigenicity in nude mice. Agene, designated MDM2, which is amplified more than 50-fold in 3T3DMcells, induced tumorigenicity when overexpressed in NIH3T3 and Rat 2cells. From the nucleotide and predicted amino acid sequence of mouseMDM2 (mMDM2), Fakharzadeh speculated that this gene encodes a potentialDNA binding protein that functions in the modulation of expression ofother genes and, when present in excess, interferes with normalconstraints on cell growth.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a method for diagnosing aneoplastic tissue, such as sarcoma, in a human.

It is another object of the invention to provide a cDNA moleculeencoding the sequence of human MDM2.

Yet another object of the invention is to provide a preparation of humanMDM2 protein which is substantially free of other human cellularproteins.

Still another object of the invention is to provide DNA probes capableof hybridizing with human MDM2 genes or mRNA molecules.

Another object of the invention is to provide antibodies immunoreactivewith human MDM2 protein.

Still another object of the invention is to provide kits for detectingamplification or elevated expression of human MDM2.

Yet another object of the invention is to provide methods foridentifying compounds which interfere with the binding of human MDM2 tohuman p53.

A further object of the invention is to provide a method of treating aneoplastic human cell.

Yet another object of the invention is to provide methods for inhibitingthe growth of tumor cells which contain a human MDM2 gene amplification.

Still another object of the invention is to provide polypeptides whichinterfere with the binding of human MDM2 to human p53.

It has now been discovered that hMDM2, a heretofore unknown human gene,plays a role in human cancer. The hMDM2 gene has been cloned and therecombinant derived hMDM2 protein shown to bind to human p53 in vitro.hMDM2 has been found to be amplified in some neoplastic cells and theexpression of hMDM2-encoded products has been found to becorrespondingly elevated in tumors with amplification of this gene. Theelevated levels of MDM2 appear to sequester p53 and allow the cell toescape from p53-regulated growth.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1 to A3, 1B-1 to B-3, and 1C-1 to C-3 show the cDNA sequence ofhuman MDM2. In this figure, human and mouse (SEQ ID NO:4)nucleotide andamino acid sequences (SEQ ID NOS:3 and 5, respectively) are compared,the mouse sequence being shown only where it differs from thecorresponding human sequence.

FIG. 2 shows that hMDM2 binds to p53.

FIGS. 3A-B illustrate the amplification of the hMDM2 gene in sarcomas.

FIGS. 4A-C illustrates hMDM2 expression.

FIGS. 5A-B show the inhibition of p53-mediated transactivation by MDM2.Yeast were stably transfected with expression plasmids encoding p53,lex-VP16, MDM2 or the appropriate vector-only controls, as indicated.p53-responsive (bars a-c) or lexA-responsive (bars d-f) β-galactosidasereporter plasmids were used to assess the response. Inset:Western blotanalysis demonstrating MDM2 (90 kD) and p53 (53 kD) expression inrepresentative yeast strains. The strain indicated by a plus wastransfected with expression vector encoding full length MDM2 and p53,while the strain indicated by a minus was transfected only with the p53expression vector.

FIG. 6 shows the determination of MDM2 and p53 domains of interaction.FIG. 5A and FIG. 5B. Random fragments of MDM2 were fused to sequencesencoding the lexA DNA binding domain and the resultant clonestransfected into yeast carrying pRS314SN (p53 expression vector) andpJK103 (exA-responsive β-galactosidase reporter). Yeast clonesexpressing β-galactosidase were identified by their blue color, and theMDM2 sequences in the lexA fusion vector were determined.β-galactosidase activity was observed independent of p53 expression inA, but was dependent on p53 expression in B. The bottom 6 clones in Bwere generated by genetic engineering. FIG. 6C. Random fragments of p53were fused to the sequence encoding the B42 acidic activation domain anda hemagglutinin epitope tag; the resultant clones were transfected intoyeast carrying lexA-MDM2 (lexA DNA binding domain fused to full lengthMDM2) and pJK103. Yeast clones were identified as above, and all werefound to be MDM2-dependent. The bottom three clones were generated bygenetic engineering.

FIGS. 7A-D show protein expression from the yeast strains described inFIG. 6. Western blot analysis was performed as described (Oliner, J. D.,et al., Nature 358:80-83 (1992)), using 20 μg of protein per lane. TheMDM2 and p53 codons contained in the fusion vectors are shown at the topof A and B, respectively. FIG. 7A. Upper panel probed with p53 Ab2detecting p53; lower panel probed with anti-lexA polyclonal antibodiesLex Ab) detecting MDM2 fusion proteins of 30-50 kD. FIG. 7B. Upper panelprobed with Lex Ab detecting the lexA-full length MDM2 fusion protein of112 kD; lower panel probed with HA Ab (a monoclonal antibody directedagainst the hemagglutinin epitope tag, Berkeley Antibody) detecting p53fusion proteins of approximately 25-30 kD.

FIGS. 8A-B show the inhibition of the p53 activation domain by MDM2.Yeast were transfected with expression vectors encoding a lexA-p53 (p53codons 1-73) fusion (bars a and b) or lexA alone (bar c). Strain b alsoexpressed full length MDM2, and all strains contained thelexA-responsive β-galactosidase reporter plasmid. Inset:Upper panelprobed with MDM2 polyclonal antibodies detecting full length MDM2 (90kD); lower panel probed with lex Ab detecting the lex-p53 fusion proteinof 40 kD.

DETAILED DESCRIPTION OF THE INVENTION

It is a discovery of the present invention that a gene exists which isamplified in some human tumors. The amplification of this gene,designated MDM2, is diagnostic of neoplasia or the potential therefor.Detecting the elevated expression of human MDM2-encoded products is alsodiagnostic of neoplasia or the potential for neoplastic transformation.Over a third of the sarcomas surveyed, including the most common boneand soft tissue forms, were found to have amplified hMDM2 sequences.Expression of hMDM2 was found to be correspondingly elevated in tumorswith the gene amplification.

Other genetic alterations leading to elevated hMDM2 expression may beinvolved in tumorigenesis also, such as mutations in regulatory regionsof the gene. Elevated expression of hMDM2 may also be involved in tumorsother than sarcomas including but not limited to those in which p53inactivation has been implicated. These include colorectal carcinoma,lung cancer and chronic myelogenous leukemia.

According to one embodiment of the invention, a method of diagnosing aneoplastic tissue in a human is provided. Tissue or body fluid isisolated from a human, and the copy number of human MDM2 genes isdetermined. Alternatively, expression levels of human MDM2 gene productscan be determined. These include protein and mRNA.

Body fluids which may be tested include urine, serum, blood, feces,saliva, and the like. Tissues suspected of being neoplastic aredesirably separated from normal appearing tissue for analysis. This canbe done by paraffin or cryostat sectioning or flow cytometry, as isknown in the art. Failure to separate neoplastic from non-neoplasticcells can confound the analysis. Adjacent non-neoplastic tissue or anynormal tissue can be used to determine a base-line level of expressionor copy number, against which the amount of hMDM2 gene or gene productscan be compared.

The human MDM2 gene is considered to be amplified if the cell containsmore than the normal copy number (2) of this gene per genome. Thevarious techniques for detecting gene amplification are well known inthe art. Gene amplification can be determined, for example, by Southernblot analysis, as described in Example 4, wherein cellular DNA from ahuman tissue is digested, separated, and transferred to a filter whereit is hybridized with a probe containing complementary nucleic acids.Alternatively, quantitative polymerase chain reaction (PCR) employingprimers can be used to determine gene amplification. Appropriate primerswill bind to sequences that bracket human MDM2 coding sequences. Othertechniques for determining gene copy number as are known in the art canbe used without limitation.

The gene product which is measured may be either mRNA or protein. Theterm elevated expression means an increase in mRNA production or proteinproduction over that which is normally produced by non-cancerous cells.Although amplification has been observed in human sarcomas, othergenetic alterations leading to elevated expression of MDM2 may bepresent in these or other tumors. Other tumors include those of lung,breast, brain, colorectal, bladder, prostate, liver, skin, and stomach.These, too, are contemplated by the present invention. Non-cancerouscells for use in determining base-line expression levels can be obtainedfrom cells surrounding a tumor, from other humans or from human celllines. Any increase can have diagnostic value, but generally the mRNA orprotein expression will be elevated at least about 3-fold, 5-fold, andin some cases up to about 100-fold over that found in non-cancerouscells. The particular technique employed for detecting mRNA or proteinis not critical to the practice of the invention. Increased productionof mRNA or protein may be detected, for example, using the techniques ofNorthern blot analysis or Western blot analysis, respectively, asdescribed in Example 4 or other known techniques such as ELISA,immunoprecipitation, RIA and the like. These techniques are also wellknown to the skilled artisan.

According to another embodiment of the invention, nucleic acid probes orprimers for the determining of human MDM2 gene amplification or elevatedexpression of mRNA are provided. The probe may comprise ribo- ordeoxyribonucleic acids and may contain the entire human MDM2 codingsequence, a sequence complementary thereto, or fragments thereof. Aprobe may contain, for example, nucleotides 1-949, or 1-2372 as shown inFIG. 1 (SEQ ID NO:2). Generally, probes or primers will contain at leastabout 14 contiguous nucleotides of the human sequence but may desirablycontain about 40, 50 or 100 nucleotides. Probes are typically labelledwith a fluorescent tag, a radioisotope, or the like to render themeasily detectable. Preferably the probes will hybridize under stringenthybridization conditions. Under such conditions they will not hybridizeto mouse MDM2(SEQ ID NO:4). The probes of the invention arecomplementary to the human MDM2 gene. This means that they share 100%identity with the human sequence.

hMDM2 protein (SEQ ID NO:3) can be produced, according to the invention,substantially free of other human proteins. Provided with the DNAsequence, those of skill in the art can express the cDNA in a non-humancell. Lysates of such cells provide proteins substantially free of otherhuman proteins. The lysates can be further purified, for example, byimmunoprecipitation, co-precipitation with p53, or by affinitychromatography.

The antibodies of the invention are specifically reactive with hMDM2protein (SEQ ID NO:3). Preferably, they do not cross-react with MDM2from other species. They can be polyclonal or monoclonal, and can beraised against native hMDM2 or a hMDM2 fusion protein or syntheticpeptide. The antibodies are specifically immunoreactive with hMDM2epitopes which are not present on other human proteins. Some antibodiesare reactive with epitopes unique to human MDM2 (SEQ ID NO:5) and notpresent on the mouse homolog. The antibodies are useful in conventionalanalyses, such as Western blot analysis, ELISA, immunohistochemistry,and other immunological assays for the detection of proteins. Techniquesfor raising and purifying polyclonal antibodies are well known in theart, as are techniques for preparing monoclonal antibodies. Antibodybinding can be determined by methods known in the art, such as use of anenzyme-labelled secondary antibody, staphylococcal protein A, and thelike. Certain monoclonal antibodies of the invention have been depositedat the American Type Culture Collection, 12301 Parklawn Drive,Rockville, Md. 20852. These include IF2, and ED9, which have beengranted accession nos. HB 11290, and HB 11291, respectively.

According to another embodiment of the invention, interference with theexpression of MDM2 provides a therapeutic modality. The method can beapplied in vivo, in vitro, or ex vivo. For example, expression may bedown-regulated by administering triple-strand forming or antisenseoligonucleotides which bind to the hMDM2 gene or mRNA, respectively, andprevent transcription or translation. The oligonucleotides may interactwith unprocessed pre-mRNA or processed mRNA. Small molecules andpeptides which specifically inhibit MDM2 expression can also be used.Similarly, such molecules which inhibit the binding of MDM2 to p53 wouldbe therapeutic by alleviating the sequestration of p53.

Such inhibitory molecules can be identified by screening forinterference of the hMDM2/p53 interaction where one of the bindingpartners is bound to a solid support and the other partner is labeled.Antibodies specific for epitopes on hMDM2 or p53 which are involved inthe binding interaction will interfere with such binding. Solid supportswhich may be used include any polymers which are known to bind proteins.The support may be in the form of a filter, column packing matrix,beads, and the like. Labeling of proteins can be accomplished accordingto any technique known in the art. Radiolabels, enzymatic labels, andfluorescent labels can be used advantageously. Alternatively, both hMDM2and p53 may be in solution and bound molecules separated from unboundsubsequently. Any separation technique known in the art may be employed,including immunoprecipitation or immunoaffinity separation with anantibody specific for the unlabeled binding partner.

It has been found that amino acid residues 13-41 of p53 (See SEQ IDNO:1) are necessary for the interaction of MDM-2 and p53. However,additional residues on either the amino or carboxy terminal side of thepeptide appear also to be required. Nine to 13 additional p53 residuesare sufficient to achieve MDM2 binding, although less may be necessary.Since cells which overexpress MDM2 escape from p53-regulated growthcontrol in sarcomas, the use of p53-derived peptides to bind to excessMDM2 leads to reestablishment of p53-regulated growth control.

Suitable p53-derived peptides for administration are those which arecircular, linear, or derivitized to achieve better penetration ofmembranes, for example. Other organic compounds which are modelled toachieve the same three dimensional structure as the peptide of theinvention can also be used.

DNA encoding the MDM2-binding, p53-derived peptide, or multiple copiesthereof, may also be administered to tumor cells as a mode ofadministering the peptide. The DNA will typically be in an expressionconstruct, such as a retrovirus, DNA virus, or plasmid vector, which hasthe DNA elements necessary for expression properly positioned to achieveexpression of the MDM2-binding peptide. The DNA can be administered,inter alia encapsulated in liposomes, or in any other form known to theart to achieve efficient uptake by cells. As in the directadministration of peptide, the goal is to alleviate the sequestration ofp53 by MDM2.

A cDNA molecule containing the coding sequence of hMDM2 (SEQ ID NO:2)can be used to produce probes and primers. In addition, it can beexpressed in cultured cells, such as E. coli, to yield preparations ofhMDM2 protein substantially free of other human proteins. The proteinsproduced can be purified, for example, with immunoaffinity techniquesusing the antibodies described above.

Kits are provided which contain the necessary reagents for determininggene copy number, such as probes or primers specific for the hMDM2 gene,as well as written instructions. The instructions can providecalibration curves to compare with the determined values. Kits are alsoprovided to determine elevated expression of mRNA (i.e., containingprobes) or hMDM2 protein (i.e., containing antibodies). Instructionswill allow the tester to determine whether the expression levels areelevated. Reaction vessels and auxiliary reagents such as chromogens,buffers, enzymes, etc. may also be included in the kits.

The human MDM2 gene has now been identified and cloned. Recombinantderived hMDM2 has been shown to bind to human p53. Moreover, it has beenfound that hMDM2 is amplified in some sarcomas. The amplification leadsto a corresponding increase in MDM2 gene products. Such amplification isassociated with the process of tumorigenesis. This discovery allowsspecific assays to be performed to assess the neoplastic or potentialneoplastic status of a particular tissue.

The following examples are provided to exemplify various aspects of theinvention and are not intended to limit the scope of the invention.

EXAMPLES Example 1

To obtain human CDNA clones, a cDNA library was screened with a murineMDM2 (mMDM2) cDNA probe. A cDNA library was prepared by usingpolyadenylated RNA isolated from the human colonic carcinoma cell lineCaCo-2 as a template for the production of random hexamer primed doublestranded cDNA. Gubler and Hoffmann, 1983, Gene 25:263-268. The cDNA wasligated to adaptors and then to the lambda YES phage vector, packaged,and plated as described by Elledge et al. (Proc. Natl. Acad. Sci. USA,88:1731-1735, 1991). The library was screened initially with aP-labelled (Kinzler, K. W., et al., Nucl. Acids Res. 17:3645-3653(1989), Feinberg and Vogelstein, 1983, Anal Biochem. 132:6-13) mMDM2cDNA probe (nucleotides 259 to 1508 (Fakharzadeh et al., 1991, EMBO J.10:1565-1569)) and then rescreened with an hMDM2 cDNA clone containingnucleotides 40 to 702.

Twelve clones were obtained, and one of the clones was used to obtainthirteen additional clones by re-screening the same library. In total,twenty-five clones were obtained, partially or totally sequenced, andmapped. Sequence analysis of the twenty-five clones revealed severalcDNA forms indicative of alternative splicing. The sequence shown inFIG. 1 (SEQ ID NO:2) is representative of the most abundant class andwas assembled from three clones:c14-2 (nucleotides 1-949), c89(nucleotides 467-1737), and c33 (nucleotides 390-2372). The 3' end ofthe untranslated region has not yet been cloned in mouse or human. The5' end is likely to be at or near nucleotide 1. There was an openreading frame extending from the 5' end of the human cDNA sequence tonucleotide 1784. Although the signal for translation initiation couldnot be unambiguously defined, the ATG at nucleotide 312 was consideredthe most likely position for several reasons. First, the sequencesimilarity between hMDM2 and mMDM2 fell off dramatically upstream ofnucleotide 312. This lack of conservation in an otherwise highlyconserved protein suggested that the sequences upstream of thedivergence may not code for protein. Second, an anchored polymerasechain reaction (PCR) approach was employed in an effort to acquireadditional upstream cDNA sequence. Ochman et al., 1985, In:PCRTechnology:Principles and Applications for DNA Amplification (Erlich,ed.) pp. 105-111 (Stockton, N.Y.). The 5' ends of the PCR derived cloneswere very similar (within 3 bp) to the 5' ends of clones obtained fromthe cDNA library, suggesting that the 5' end of the hMDM2 sequence shownin FIG. 1 may represent the 5' end of the transcript. Third, in vitrotranslation of the sequence shown in FIG. 1, beginning with themethionine encoded by the nucleotide 312 ATG, generated a proteinsimilar in size to that observed in human cells.

In FIG. 1, hMDM2 CDNA sequence (SEQ ID NO:2) hMDM2 (SEQ ID NO:3) andmMDM2 (SEQ ID NO:4) nucleotide and amino acid sequences (SEQ ID NO:5)are compared. The mouse sequence is only shown where it differs from thecorresponding human sequence. Asterisks mark the 5' and 3' boundaries ofthe previously published mMDM2 cDNA. Fakharzadeh et al., 1991, EMBO J.10:1565-1569. Dashes indicate insertions. The mouse and human amino acidsequences are compared from the putative translation start site atnucleotide 312 through the conserved stop codon at nucleotide 1784.

Comparison of the human and mouse MDM2 coding regions revealedsignificant conservation at the nucleotide (80.3%) and amino acid(80.4%) levels. Although hMDM2 and mMDM2 bore little similarity to othergenes recorded in current databases, the two proteins shared severalmotifs. These included a basic nuclear localization signal (Tanaka,1990, FEBS Letters 271:41-46) at codons 181 to 185, several caseinkinase II serine phosphorylation sites (Pinna, 1990, Biochem. et.Biophys. Acta. 1054:267-284) at codons 166 to 169, 192 to 195, 269 to272, and 290 to 293, an acidic activation domain (Ptashne, 1988, Nature355:683-689) at codons 223 to 274, and two metal binding sites(Harrison, 1991, Nature 353:715) at codons 305 to 322 and 461 to 478,neither of which is highly related to known DNA binding domains. Theprotein kinase A domain noted in mMDM2 (Fakharzadeh et al., 1991, EMBOJ. 10:1565-1569) was not conserved in hMDM2.

Example 2

To determine whether the hMDM2 protein could bind to human p53 proteinin vitro, an hMDM2 expression vector was constructed from the cDNAclones. The hMDM2 expression vector was constructed in pBluescript SK+(Stratagene) from overlapping cDNA clones. The construct contained thesequence shown in FIG. 1 (SEQ ID NO:2) from nucleotide 312 to 2176. A 42bp black bettle virus ribosome entry sequence (Dasmahapatra et al.,1987, Nucleic Acid Research 15:3933) was placed immediately upstream ofthis hMDM2 sequence in order to obtain a high level of expression. Thisconstruct, as well as p53 (El-Deriy et al., 1992, Nature Genetics, inpress) and MCC (Kinzler et al., 1991, Science 251:1366-1370) constructsin pBluescript SK+, were transcribed with T7 RNA polymerase andtranslated in a rabbit reticulocyte lysate (Promega) according to themanufacturer's instructions.

Although the predicted size of the protein generated from the constructwas only 55.2 kd (extending from the methionine at nucleotide 312 tonucleotide 1784), in vitro translated protein migrated at approximately95 kilodaltons.

Ten μl of lysate containing the three proteins (hMDM2, p53 and MCC),alone or mixed in pairs, were incubated at 37° C. for 15 minutes. Onemicrogram (10 μl) of p53 Ab1 (monoclonal antibody specific for theC-terminus of p53) or Ab2 (monoclonal antibody specific for theN-terminus of p53) (Oncogene Science), or 5 μl of rabbit serumcontaining MDM2 Ab (polyclonal rabbit anti-hMDM2 antibodies) orpreimmune rabbit serum (obtained from the rabbit which produced thehMDM2 Ab), were added as indicated. The polyclonal rabbit antibodieswere raised against an E. coli-produced hMDM2-glutathione S-transferasefusion protein containing nucleotides 390 to 816 of the hMDM2 cDNA.Ninety μl of RIPA buffer (10 mM tris pH 7.5 !, 1% sodium deoxycholate,1% NP40, 150 mM NaCl, 0.1% SDS), SNNTE buffer, or Binding Buffer(El-Deriy et al., 1992, Nature Genetics, in press) were then added andthe mixtures allowed to incubate at 4° C. for 2 hours.

Two milligrams of protein A sepharose were added to each tube, and thetubes were rotated end-over-end at 4° C. for 1 hour. After pelleting andwashing, the immunoprecipitates were subjected to SDS-polyacrylamide gelelectrophoresis and the dried gels autoradiographed for 10 to 60 minutesin the presence of Enhance (New England Nuclear).

FIG. 2 shows the co-precipitation of hMDM2 and p53. The three buffersproduced similar results, although the co-precipitation was lessefficient in SNNTE buffer containing 0.5 M NaCl (FIG. 2, lanes 5 and 8)than in Binding Buffer containing 0.1 M NaCl (FIG. 2 lanes 6 and 9).

In vitro translated hMDM2, p53 and MCC proteins were mixed as indicatedabove and incubated with p53 Ab1, p53 Ab2, hMDM2 Ab, or preimmune serum.Lanes 1, 4, 7, 10 and 14 contain aliquots of the protein mixtures usedfor immunoprecipitation. The bands running slightly faster than p53 arepolypeptides produced from internal translation initiation sites.

The hMDM2 protein was not immunoprecipitated with monoclonal antibodiesto either the C-terminal or N-terminal regions of p53 (FIG. 2, lanes 2and 3). However, when in vitro translated human p53 was mixed with thehMDM2 translation product, the anti-p53 antibodies precipitated hMDM2protein along with p53, demonstrating an association in vitro (FIG. 2,lanes 5 and 6). As a control, a protein of similar electrophoreticmobility from another gene (MCC (Kinzler et al., 1991, Science251:1366-1370)) was mixed with p53. No co-precipitation of the MCCprotein was observed (FIG. 2, lanes 8 and 9). When an in vitrotranslated mutant form of p53 (175^(his)) was mixed with hMDM2 protein,a similar co-precipitation of hMDM2 and p53 proteins was also observed.

In the converse of the experiments described above, the anti-hMDM2antibodies immunoprecipitated p53 when mixed with hMDM2 protein (FIG. 2,lane 15) but failed to precipitate p53 alone (FIG. 5, lane 13).Preimmune rabbit serum failed to precipitate either hMDM2 or p53 (FIG.2, lane 16).

Example 3

In order to ascertain the chromosomal localization of hMDM2, somaticcell hybrids were screened with an hMDM2 cDNA probe. A human-hamsterhybrid containing only human chromosome 12 was found to hybridize to theprobe. Screening of hybrids containing portions of chromosome 12(Turc-Carel et al., 1986, Cancer Genet. Cytogenet. 23:291-299) with thesame probe narrowed the locaization to chromosome 12q12-14.

Example 4

Previous studies have shown that this region of chromosome 12 is oftenaberrant in human sarcomas. Mandahl et al., 1987, Genes Chromosomes &Cancer 1:9-14; Turc-Carel et al., 1986, Cancer Genet. Cytogenet.23:291-299; Meltzer et al., 1991, Cell Growth & Differentiation2:495-501. To evaluate the possibility that hMDM2 was geneticallyaltered in such cancers, Southern blot analysis was performed.

FIG. 3 shows examples of the amplification of the hMDM2 gene insarcomas. Cellular DNA (5 μg) was digested with EcoRI, separated byagarose gel electrophoresis, and transferred to nylon as described byReed and Mann (Nucl. Acids Res., 1985, 13:7207-7215). The cellular DNAwas derived from five primary sarcomas (lanes 1-4, 6) and one sarcomacell line (OsA-C1, lane 5). The filters were then hybridized with anhMDM2 cDNA fragment probe nucleotide 1-949 (see FIG. 1, SEQ ID NO:2), orto a control probe which identifies fragments of similar size (DCC gene,1.65 cDNA fragment). Fearon, 1989, Science 247:49-56. Hybridization wasperformed as described by Vogelstein et al. (Cancer Research, 1987,47:4806-4813). A striking amplification of hMDM2 sequences was observedin several of these tumors. (See FIG. 3, lanes 2, 3 and 5). Of 47sarcomas analyzed, 17 exhibited hMDM2 amplification ranging from 5 to 50fold. These tumors included 7 to 13 liposarcomas, 7 of 22 malignantfibrous histiocytomas (MFH), 3 of 11 osteosarcomas, and 0 and 1rhabdomyosarcomas. Five benign soft tissue tumors (lipomas) andtwenty-seven carcinomas (colorectal or gastric) were also tested bySouthern blot analysis and no amplification was observed. Example 5

This example illustrates that gene amplification is associated withincreased expression.

FIG. 4A illustrates hMDM2 expression as demonstrated by Northern blotanalysis. Because of RNA degradation in the primary sarcomas, only thecell lines could be productively analyzed by Northern blot. RNA wasseparated by electrophoresis in a MOPS-formaldehyde gel andelectrophoretically transferred to nylon filters. Transfer andhybridization were performed as described by Kinzler et al. (Nature332:371-374, 1988). The RNA was hybridized to the hMDM2 fragmentdescribed in FIG. 3. Ten μg of total RNA derived, respectively, from twosarcoma cell lines (OsA-CL, lane 1 and RC13, lane 2) and the colorectalcancer cell line (CaCo-2) used to make the cDNA library (lane 3). Lane 4contains 10 μg of polyadenylated CaCo-2 RNA. RNA sizes are shown in kb.In the one available sarcoma cell line with hMDM2 amplification, asingle transcript of approximately 5.5 kb was observed (FIG. 4A, lane1). The amount of this transcript was much higher than in a sarcoma cellline without amplification (FIG. 4A, lane 2) or in a carcinoma cell line(FIG. 4A, lane 3). When purified mRNA (rather than total RNA) from thecarcinoma cell line was used for analysis, an hMDM2 transcript of 5.5 kbcould also be observed (FIG. 4A, lane 4).

FIG. 4B illustrates hMDM2 expression as demonstrated by Western blotanalysis of the sarcoma cell lines RC13 (lane 1), OsA-CL (lane 3), HOS(lane 4), and the carcinoma cell line CaCo-2 (lane 2).

FIG. 4C illustrates hMDM2 expression as demonstrated by Western blotanalysis of primary sarcomas. Lanes 1 to 3 contain protein from sarcomaswith hMDM2 amplifications, and lanes 4 and 5 contain protein fromsarcomas without hMDM2 amplification.

Western blots using affinity purified MDM2 Ab were performed with 50 μgprotein per lane as described by Kinzler et al. (Mol. Cell. Biol, 1990,10:634-642), except that the membranes were blocked in 10% nonfat driedmilk and 10% goat serum, and secondary antibodies were coupled tohorseradish peroxidase, permitting chemiluminescent detection (AmershamECL). MDM2 Ab was affinity purified with a pATH-hMDM2 fusion proteinusing methods described in Kinzler et al. (Mol Cell. Biol. 10:634-642,1990). Non-specifically reactive proteins of about 75-85, 105-120 and170-200 kd were observed in all lanes, irrespective of hMDM2amplification status. hMDM2 proteins, of about 90-97 kd, were observedonly in the hMDM2-amplified tumors. Protein marker sizes are shown inkd.

A protein of approximately 97 kilodaltons was expressed at high levelsin the sarcoma cell line with hMDM2 amplification (FIG. 4B, lane 3),whereas no expression was evident in two sarcoma cell lines withoutamplification or in the carcinoma cell line (FIG. 4B, lanes 1, 2 and 4).Five primary sarcomas were also examined by Western blot analysis. Threeprimary sarcomas with amplification expressed the same size protein asthat observed in the sarcoma cell line (FIG. 4C, lanes 1-3), while noprotein was observed in the two sarcomas without amplification (FIG. 4C,lanes 4 and 5).

Expression of the hMDM2 RNA in the sarcoma with amplification wasestimated to be at least 30 fold higher than that in the other linesexamined. This was consistent with the results of Western blot analysis.

The above examples demonstrate that hMDM2 binds to p53 in vitro and isgenetically altered (i.e., amplified) in a significant fraction ofsarcomas, including MFH, liposarcomas, and osteosarcomas. These are themost common sarcomas of soft tissue and bone. Weiss and Enzinger, 1978,Cancer 41:2250-2266; Malawer et al., 1985, In: Cancer:Principles andPractice of Oncology, DeVita et al., Eds., pp. 1293-1342 (Lippincott,Philadelphia).

Human MDM2 amplification is useful for understanding the pathogenesis ofthese often lethal cancers.

MDM2 may functionally inactivate p53 in ways similar to those employedby virally encoded oncoproteins such as SV40 T-antigen, adenovirus E1B,and HPV E6. Lane and Bechimol, 1990, Genes and Developmet 4:1-8; Wernesset al., 1990, Science 248:76. Consistent with this hypothesis, nosarcomas with hMDM2 amplification had any of the p53 gene mutations thatoccur commonly in other tumors. hMDM2 amplification provides a parallelbetween viral carcinogenesis and the naturally occurring geneticalterations underlying sporadic human cancer. The finding thatexpression of hMDM2 is correspondingly elevated in tumors withamplification of the gene are consistent with the finding that MDM2binds to p53, and with the hypothesis that overexpression of MDM2 insarcomas allows escape from p53 regulated growth control. This mechanismof tumorigenesis has striking parallels to that previously observed forvirally induced tumors (Lane and Bechimol, 1990, Genes and Development4:1-8; Werness et al., 1990, Science 248:76), in which viral oncogeneproducts bind to and functionally inactivate p53.

Example 6

This example demonstrates that MDM2 expression inhibits p53-mediatedtransactivation.

To determine if MDM2 could influence the ability of p53 to activatetranscription, expression vectors coding for the two proteins werestably transfected into yeast along with a p53-responsive reporterconstruct. The reporter consisted of a β-galactosidase gene under thetranscriptional control of a minimal promoter and a multimerized humanDNA sequence which strongly bound p53 in vitro (Kern, S. E., et al.,Science 256:827-830 (1992). Reporter expression was completely dependenton p53 in this assay (FIG. 5, compare bars a and c). MDM2 expression wasfound to inhibit p53-mediated transactivation of this reporter 16foldrelative to isogeneic yeast lacking MDM2 expression (FIG. 5, comparebars a and b). Western blot analysis confirmed that p53 (53 kD) wasexpressed equivalently in strains with and without MDM2 (90 kD) (FIG. 1,inset).

METHODS. The MDM2 expression plasmid, pPGK-MDM2, was constructed byinserting the full length MDM2 cDNA (Oliner, J. D., et al., Nature358:80-83 (1992)) into pPGK (Poon, D. et aL, Mol. and Cell. Biol1111:4809-4821 (1991)), immediately downstream of the phosphoglyceratekinase constitutive promoter. Galactose-inducible p53 (pRS314SN, Nigro,J. M., et al., Mol. and Cell Biol. 12:1357-1365 (1992)), lexA-VP16(YVLexA, Dalton, S., et al., Cell 68:597-612 (1992)), and lexA (YLexA,YVLexA minus VP16) plasmids were used as indicated. The reporters werePG16-lacZ (Kern, S. E. et al., Science 256:827-830 (1992))(p53-responsive) and pJK103 (Kamens, J., et al., Mol. Cell. Biol.10:2840-2847 (1990)) LexA-responsive). S. cerevisiae strain pEGY48 wastransformed as described (Kinzler, K. W. et al., Nucl. Acids Res.17:36453-653 (1989)). Yeast strains represented by bars a-c were grownat 30° C. to mid-log phase in selective liquid medium containing 2%raffinose as the carbon source, induced for 30 minutes by the additionof 2% galactose, harvested, and lysed as described (Kern, S.E. et al.,Science 256:827-830 (1992)). The strains represented by bars d-f weretreated similarly, except that the cells were induced in galactose for 4hours to obtain measurable levels of β-galactosidase. β-galactosidaseactivities shown represent the mean of three to five experimental values(error bars indicate s.e.m.). Protein concentrations were determined bya Coomassie blue-based (bio-Rad) assay. The β-galactosidase assays wereperformed with AMPGD chemiluminescent substrate and Emerald enhancerTropix) according to the manufacturer's instructions. β-glactosidaseactivities of bars b and c are shown relative to that of bar A;β-galactosidase activities of bars e and f are shown relative to that ofbar d. Western blots were performed as described (Oliner, J. D., et al.,Nature 358:80-83 (1992)), using p53 Ab1801 (lower panel, OncogeneScience) or MDM2 polyclonal antibodies (Oliner, J. D., et al., Nature358:80-83 (1992)) (upper panel).

To ensure that this inhibition was not simply a general transcriptionaldown regulation mediated by the expression of the foreign MDM2 gene, ayeast strain was created that contained a different transcriptionalactivator lexA-VP16, consisting of the lexA DNA binding domain fused tothe VP16 acidic activation domain), a similar reporter (with alexA-responsive site upstream of a β-galactosidase gene), and the sameMDM2 expression vector. The results shown in FIG. 1 (bars d & e)demonstrate that lexA-VP16 transactivation was unaffected by thepresence of MDM2. Furthermore, MDM2 expression had no apparent effect onthe growth rate of the cells.

Example 7

This example demonstrates the domains of p53 and MDM2 which interactwith each other.

To gain insight into the mechanism of the MDM2-mediated p53 inhibition,the domains of MDM2 and p53 responsible for binding to one another weremapped. The yeast system used to detect protein-protein binding takesadvantage of the modular nature of transcription factor domains (Keegan,L., et al., Science 231:699-704 (1986); Chien, C.-T., Proc. Natl. Acad.Sci. U.S.A. 88:9578-9582 (1991); Brent, R., et al., Cell 43:729-731(1985); Ma, J., et al., Cell 55:4430446 (1988). Generically, if protein1 (fused to a sequence-specific DNA binding domain) is capable ofbinding to protein 2 (fused to a transcriptional activation domain),then co-expression of both fusion proteins will result intranscriptional activation of a suitable reporter. In our experiments,the lexA DNA binding domain (amino acids 2-202) and the B42 acidicactivation domain (AAD) were used in the fusion constructs. The reporter(Kamens, J., et al., Mol Cell. Biol. 10:2840-2847 (1990); contained alexA-responsive site upstream of a β-galactosidase gene. As an initialcontrol experiment, full length MDM2 was inserted into the lexA fusionvector, and full length p53, supplying its intrinsic activation domainwas inserted into a non-fusion vector. The combination resulted in theactivation of the lexA-responsive reporter, while the same expressionconstructs lacking either the MDM2 or p53 cDNA inserts failed toactivate β-galactosidase (Table I, strains 1, 2, and 3). Thus,activation was dependent upon MDM2-p53 binding.

This assay was then applied to mapping the interaction domains of eachprotein. Full length cDNA fragments encoding MDM2 or p53 were randomlysheared by sonication, amplified by polymerase chain reaction, sizefractionated, cloned into the appropriate fusion vectors and transfectedinto yeast along with the reporter and the full length version of theother protein. METHODS. Full length MDM2 cDNA in pBluescriptSK+(Stratagene) was digested with XhoI and BamHI to excise the entireinsert. After agarose gel purification, the insert was sheared intorandom fragments by sonication, polished with the Klenow fragment of DNApolymerase I, ligated to catch linkers, and amplified by the polymerasechain reaction as described (Kinzler, K.W., et al., Nucl. Acids Res.17:3645-3653 (1989)). The fragments were fractionated on an acrylamidegel into size ranges of 100-400 bp or 400-1000 pb, cloned intolexA(1-202)+PL (Ruden, D. M., et al., Nature 350:250-252 (1991)), andtransfected into bacteria (XL-1 Blue, Stratagene). At least 10,000bacterial colonies were scraped off agar plates, and the plasmid DNA wastransfected into a strain of pEGY48 containing pRS314N (p53 expressionvector) and pJK103 lexA-responsive β-galactosidase reporter).Approximiately 5,000 yeast clones were plated on selective mediumcontaining 2% dextrose, and were replica-plated onto glalctose- andX-gal-containing selective medium. Blue colonies (17) appeared only onthe plates containing the larger fragments of MDM2. The 17 isolatedcolonies were tested for blue color in this assay both in the presenceand in the absence of galactose (p53 induction); all tested positive inthe presence of galactose but only 2 of the 17 tested positive in itsabsence. MDM2-containing plasmid DNA extracted from the 17 yeast cloneswas selectively transferred to bacterial strain KC8 and sequenced fromthe lexA-MDM2 junction. The MDM2 sequences of the two p53-independentclones are diagrammed in FIG. 6A. The MDM2 sequences of the remaining 15p53-dependent clones coded for peptides ranging from 135 to 265 a.a. inlength and began exclusively at the initiator methionine. Three of theMDM2 sequences obtained are shown at the top of FIG. 6B. The lower 6sequences were genetically engineered (using the polymerase chainreaction and appropriate primers) into lexA(1-202)+PL and subsequentlytested to further narrow the binding region.

Fragments of p53 were also cloned into pJG4-5, producing a fusionprotein C-terminal to the B42 acidic activation domain and incorporatingan epitope of hemagglutinin. The clones were transfected into a strainof pEGY48 already containing lex-MDM2 (plex-202+PL containing fulllength MDM2) and pJK103. The top three p53 sequences shown in FIG. 6C.were derived from yeast obtained by colony screening, whereas the lowerthree were genetically engineered to contain the indicated fragments.

The resultant yeast colonies were examined for β-galactosidase activityin situ. Of approximately 5000 clones containing MDM2 fragments fused tothe lexA DNA binding domain, 17 were found to score positively in thisassay. The clones could be placed into two classes. The first class (twoclones) expressed low levels of β-galactosidase (about 5-fold less thanthe other fifteen clones) and β-galactosidase expression was independentof p53 expression (FIG. 6A). These two clones encoded MDM2 amino acids190-340 and 269-379, respectively. The region shared between these twoclones overlapped the only acidic domain in MDM2 (amino acids 230-301).This domain consisted of 37.5% aspartic and glutamic acid residues butno basic amino acids. This acidic domain appears to activatetranscription only when isolated from the rest of the MDM2 sequence,because the entire MDM2 protein fused to lexA had no measurableβgalactosidase activity in the same assay (Table I, strain 3). The otherclass (15 clones) each contained the amino terminal region of MDM2 (FIG.6B). The β-galactosidase activity of these clones was dependent on p53co-expression. To narrow down the region of interaction, we generatedsix additional clones by genetic engineering. The smallest tested regionof MDM2 which could functionally interact with full length p53 containedMDM2 codons 1 to 118 (FIG. 6B). The relatively large size of the domainrequired for interaction was consistent with the fact that when smallsonicated fragments of MDM2 were used in the screening assay (200 bpinstead of 600 bp average size), no positively scoring clones wereobtained.

In a converse set of experiments, yeast clones containing fragments ofp53 fused to the B42 AAD were screened for lexA-responsive reporterexpression in the presence of a lexA-MDM2 fusion protein. Sequencing ofthe 14 clones obtained in the screen revealed that they could be dividedinto three subsets, one containing amino acids 1-41, a second containingamino acids 13-57, and a third containing amino acids 1-50 (FIG. 2C);SEQID NO:1. The minimal overlap between these three fragments containedcodons 13-41. Although this minimal domain was apparently necessary forinteraction with MDM2, it was insufficient, as the fragments required9-12 amino acids on either side of codons 13-41 for activity (FIG. 6C).To further test the idea that the amino termainal region of p53 wasrequired for MDM2 binding, we generated an additional yeast strainexpressing the lex-DNA binding domain fused to p53 codons 74-393) andthe B42 acidic activation domainfused to full length MDM2. These strainsfailed to activate the same lexA-responsive reporter (Table I, strain8), as expected if the N-terminus of p53 were required for theinteraction.

                  TABLE I                                                         ______________________________________                                        STRAIN p53          MDM2                                                      NUMBER CONSTRUCT    CONSTRUCT     ACTIVATION                                  ______________________________________                                        1      p53.sup.a    Vector.sup.b  -                                           2      p53.sup.a    lexA-MDM2.sup..sup.b                                                                        +                                           3      Vector.sup.a lexA-MDM2.sup.b                                                                             -                                           4      p53.sup.a    lexA-MDM2 (1-118).sup.b                                                                     +                                           5      Vector.sup.a lexA-MDM2 (1-118).sup.b                                                                     -                                           6      B42-p53 (141).sup.c                                                                        lexA-MDM2.sup.b                                                                             +                                           7      B42-p53 (141).sup.c                                                                        Vector.sup.b  -                                           8      lexA-p53 (74-393).sup..sup.b                                                               B42-MDM2.sup.c                                                                              -                                           9      p53 (1-137).sup.a                                                                          lexA-MDM2.sup.b                                                                             -                                           ______________________________________                                         The MDM2 and p53 proteins expressed in each strain, along with the            relevant reporters, are indicated. Numbers in parentheses refer to the        MDM2 or p53 amino acids encoded (absence of parentheses indicated full        length protein, that is, MDM2 amino acids 1 to 491 or p53 amino acids 1 t     393). The lexA responsive β-galactosidase reporter plasmid (pJK103,      Kamens, J., et al., Mol. Cell. Biol. 10:2840-2847 (1990)) was present in      all strains.                                                                  .sup.a PRS314 vector (Nigro, J. M., et al., Mol. and Cell. Biol.              12:1357-1365 (1992).                                                          .sup.b plex(1-202) +PL vector, containing lexA DNA binding domain fused t     insert (Ruden, D. M., et al., Nature 350:250-252 (1991).                      .sup.c pJG45 vector, containing B42 activation domain fused to insert.        .sup.d (+) indicates that colonies turned blue following 24 hours of          incubation on Xgal-containing selective medium, while (-) indicates that      colonies remained white following 72 hours of incubation.                

Sequence analysis showed that all p53 and MDM2 fragments noted in FIG. 6were ligated in frame and in the correct orientation relative to the B42and lexA domains, respectively. Additionally, all clones compared inFIG. 6 expressed the relevant proteins at similar levels, as shown byWestern blotting (FIG. 7).

The most striking results of these mapping experiments was that theregion of p53 required to bind MDM2 was almost identical to thepreviously identified acidic activation domain of p53 (amino acids20-42) (Unger, T., et al., EMBO J. 11:1383-1390 (1992); Miller, C. W.,et al., Proc. Am. Assoc. Cancer Res. 33:386 (1992). This suggested thatMDM2 inhibits p53-mediated transcriptional activation by "concealing"the activation domain of p53 from the transcriptional machinery. If thiswere true, the p53 activation domain, in isolation from the rest of thep53 protein, should still be inhibitable by full length MDM2. To testthis hypothesis, we produced a hybrid protein containing the p53activation domain (codons 1-73) fused to the lexA-DNA binding domain.This construct exhibited strong transcriptional activation of alexA-responsive reporter (FIG. 8), as predicted from previousexperiments in which the p53 activation domain was fused to another DNAbinding domain (Fields, S., et al., Science 249:1046-1049 (1990);Raycroft, L., et al., Science 249:1049-1051 (1990)). The lexA-p53 DNAconstruct was stably expressed in yeast along with the full length MDM2expression vector (or the vector alone). MDM2 expression resulted in afive-fold decrease in reporter activity, demonstrating that MDM2 canspecifically inhibit the function of the p53 activation domainregardless of the adjacent protein sequences tethering p53 to DNA (FIG.8).

METHODS. Strains were grown to mid-log phase in 2% dextrose beforeinduction of p53 expression for 2 hours by the addition of 2% galactose.

The lex-p53 construct was identical to lex-VP16 (YVlexA, Dalton, S., etal., Cell 68:597-612 (1992)) except that VP16 sequences were replaced byp53 sequences encoding amino acids 1 to 73.

The results obtained in the experiments discussed herein raise aninteresting paradox. If MDM2 binds to (FIG. 6) and conceals (FIG. 8) thep53 activation domain from the transcriptional machinery, how could thelexA-MDM2-p53 complex activate transcription from the lexA-responsivereporter (Table I, strain 2)? Because the only functional activationdomain in the lexA-MDM2-p53 complex of strain 2 is expected to becontributed by p53, one might predict that it would be concealed bybinding to MDM2 and thereby fail to activate. A potential resolution ofthis paradox is afforded by knowledge that p53 exists as a homotetramer(Stenger, J.E., et al., Mol. Carcinogenesis 5:102-106 (1992);Sturzbecher, H. W. et al., Oncogene 7:1513-1523 (1992). Thus theactivation noted in the lexA-MDM2-p53 complex could be due to thepresence of four individual activation domains contributed by the p53tetramer, not all of which were concealed by MDM2. As a direct test ofthis issue, the domain of p53 required for homo-oligomerization(Stenger, J. E., et al., Mol. Carcinogenesis 5:102-106 (1992);Sturzbecher, H. W. et al, Oncogene 7:1513-1523 (1992) (the C-terminus)was removed from the p53 expression construct, so that it consisted ofonly codons 1-137. This truncated p53 polypeptide retained the entireactivation domain (as shown in FIG. 8, bar a) and the entire domainrequired for interaction with MDM2 (Table I, strain 6). Yet, whenallowed to interact with lexA-MDM2, no transactivation of thelexA-responsive reporter was observed (Table I, strain 9). Because p53did not inhibit lexA-MDM2 binding to the lexA reporter (Table I, strain2), the result of strain 9 is likely to be due to a direct inhibition ofthe isolated p53 activation domain by MDM2.

Example 8

This example illustrates the production and characterization ofantibodies specific for MDM2 epitopes.

The antigen preparations used to intraperitoneally immunize female(BALB/c×C57BL/6)F1 mice comprised bacterially expressed,glutathione-column purified glutathione-S-transferase-MDM2 (GST-MDM2)fusion protein. (One preparation was further purified on apolyacrylamide gel and electroeluted.) The fusion protein contains a 16kD amino terminal portion of human MDM2 protein (amino acids 27 to 168;SEQ ID NO:3. For immunization, the fusion protein was mixed with Ribiadjuvant (Ribi Immunochem Research, Inc.).

Two mice were sacrificed and their spleen cells fused to SP2/0s myelomacells (McKenzie, et al., Oncogene, 4:543-548, 1989). Resultinghybridomas were screened by ELISA on trpE-MDM2 fusion protein-coatedmicrotiter wells. The trpE-MDM2 fusion protein contains the same portionof MDM2 as the GST-MDM2 fusion protein. Antigen was coated at aconcentration of 1 μg/ml.

A second fusion was performed as described except hybridomas werescreened on electroeluted, glutathione purified GST-MDM2. Positivehybridomas from both fusions were expanded and single cell subcloned.Subclones were tested by Western Blot for specificity to the 55 kDtrpE-MDM2 and the 43 kD GST-MDM2 fusion proteins.

Two Western Blot positive subclones (1F2 and JG3) were put into mice forascites generation. The resulting ascites were protein A purified. Bothpurified monoclonal antibodies tested positive by Western Blot andimmunoprecipitation for the 90 KD migrating MDM2 protein present in ahuman osteosarcoma cell line (OsA-CL), which overexpresses MDM2, andnegative in the HOS osteosarcoma, which does not overexpress MDM2.

ED9 was protein G-purified from ascites and found to be specific incryostat immunohistochemistry for MDM2 in osteosarcoma cells, as wasIF2.

Example 9

This example demonstrates the expression and detection of MDM2 at thecellular level.

To evaluate MDM2 expression at the cellular level, we producedmonoclonal antibodies against bacterially generated fusion proteinscontaining residues 27 to 168 of MDM2. (See example 8.) Of severalantibodies tested, mAb IF-2 was the most useful, as it detected MDM2 inseveral assays. For initial testing, we compared proteins derived fromOsA-CL, a sarcoma cell line with MDM2 amplification but without p53mutation (Table II) and proteins from SW480, a colorectal cancer cellline with p53 mutation (Barak et al., EMBO 12:461-468 (1993)) butwithout MDM2 amplification (data not shown). We could not distinguishwhether the low molecular weight bands in OsA-CL were due to proteindegradation or alternative processing of MDM2 transcripts. The more than20-fold difference in intensity between the signals observed in OsA-CLand SW480 is consistent with the greater than 20-fold difference in MDM2gene copy number in these two lines. Conversely, the 53 kd signaldetected with p53-specific mAb 1801 was much stronger in SW480 than inOsA-CL consistent with the presence of a mutated p53 in SW480

Cells grown on cover slips were then used to assess the cellularlocalization of the MDM2 protein. A strong signal, exclusively nuclear,was observed in OsA-CL cells with the IF-2 mAb and a weaker signal,again strictly nuclear, was observed in SW480 The nuclear localizationof MDM2 is consistent with previous studies of mouse cells (Barak etal., EMBO 12:461-468 (1993)) and the fact that human MD2 contains anuclear localization signal at residues 179 to 186. Reactivity with thep53-specific antibody was also confined to the nuclei of these two celllines with the relative intensities consistent with the Western blotresults.

The IF-2 mAb was then used (at 5 μg/ml) to stain the seven primarysarcomas noted above. The nuclei of two of them (tumors #3 and #10)stained strongly. Both of these tumors contained MDM2 gene amplification(Table II). In the five tumors without amplification, little or no MDM2reactivity was observed.

                                      TABLE II                                    __________________________________________________________________________    TUMOR                                                                              TUMOR        MDM2     p53            OVER-                               #    ID   TYPE.sup.a                                                                            AMPLIFICATION.sup.b                                                                    ALTERATION.sup.c                                                                             EXPRESSION.sup.d                    __________________________________________________________________________    1    M-2  MFH     ABSENT   DELETION/      NONE                                                           REARRANGEMENT                                      2    M-5  MFH     ABSENT   CGC-CUC MUTATION;                                                                            p53                                                            Arg(158)-His                                       3    M-7  MFH     PRESENT  NONE OBSERVED  MDM2                                4    M-8  MFH     ABSENT   DELETION       NONE                                5    M-14 MFH     ABSENT   NONE OBSERVED  N.T.                                6    M-15 MFH     ABSENT   DELETION       N.T.                                7    M-16 MFH     ABSENT   NONE OBSERVED  NONE                                8    M-17 MFH     ABSENT   NONE OBSERVED                                      9    M-18 MFH     ABSENT   OVEREXPRESSED  p53                                 10   M-20 MFH     PRESENT  NONE OBSERVED  MDM2                                11   L-5  LIPOSARCOMA                                                                           ABSENT   NONE OBSERVED  N.T.                                12   L-7  LIPOSARCOMA                                                                           ABSENT   AAC-AGC MUTATION;                                                                            N.T.                                                           Asn(239)-Ser                                       13   L-9  LIPOSARCOMA                                                                           PRESENT  NONE OBSERVED  N.T.                                14   L-11 LIPOSARCOMA                                                                           ABSENT   NONE OBSERVED  N.T.                                15   KL5B LIPOSARCOMA                                                                           ABSENT   CAG-UAG MUTATION;                                                                            N.T.                                                           Gln(144)-Stop                                      16   KL7  LIPOSARCOMA                                                                           PRESENT  NONE OBSERVED  N.T.                                17   KL10 LIPOSARCOMA                                                                           ABSENT   NONE OBSERVED  N.T.                                18   KL11 LIPOSARCOMA                                                                           ABSENT   GGT-GAT MUTATION; EXON 5                                                                     N.T.                                                           SPLICE DONOR SITE                                  19   KL12 LIPOSARCOMA                                                                           ABSENT   NONE OBSERVED  N.T.                                20   KL28 LIPOSARCOMA                                                                           PRESENT  NONE OBSERVED  N.T.                                21   KL30 LIPOSARCOMA                                                                           PRESENT  NONE OBSERVED                                      22   S189 LIPOSARCOMA                                                                           PRESENT  NONE OBSERVED  N.T.                                23   S131B                                                                              LIPOSARCOMA                                                                           ABSENT   NONE OBSERVED  N.T.                                24   OSA-CL                                                                             MFH     PRESENT  NONE OBSERVED  MDM2                                __________________________________________________________________________     .sup.a MFH = malignant fibrous histiocytoma                                   .sup.b as assessed by Southern blot                                           .sup.c as assessed by Southern blot, sequencing of exons 5-8, or              immunohistochemical analysis                                                  .sup.d as assessed by immunohistochemical analysis; N.T. = not tested    

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 9                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 64 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (viii) POSITION IN GENOME:                                                    (A) CHROMOSOME/SEGMENT: 17q                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       MetGluGluProGlnSerAspProSerValGluProProLeuSerGln                              151015                                                                        GluThrPheSerAspLeuTrpLysLeuLeuProGluAsnAsnValLeu                              202530                                                                        SerProLeuProSerGlnAlaMetAspAspLeuMetLeuSerProAsp                              354045                                                                        AspIleGluGlnTrpPheThrGluAspProGlyProAspGluAlaPro                              505560                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2372 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (H) CELL LINE: CaCo-2                                                         (viii) POSITION IN GENOME:                                                    (B) MAP POSITION: 12q12-14                                                    (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 312..1784                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GCACCGCGCGAGCTTGGCTGCTTCTGGGGCCTGTGTGGCCCTGTGTGTCGGAAAGATGGA60                GCAAGAAGCCGAGCCCGAGGGGCGGCCGCGACCCCTCTGACCGAGATCCTGCTGCTTTCG120               CAGCCAGGAGCACCGTCCCTCCCCGGATTAGTGCGTACGAGCGCCCAGTGCCCTGGCCCG180               GAGAGTGGAATGATCCCCGAGGCCCAGGGCGTCGTGCTTCCGCAGTAGTCAGTCCCCGTG240               AAGGAAACTGGGGAGTCTTGAGGGACCCCCGACTCCAAGCGCGAAAACCCCGGATGGTGA300               GGAGCAGGCAAATGTGCAATACCAACATGTCTGTACCTACTGATGGTGCT350                         MetCysAsnThrAsnMetSerValProThrAspGlyAla                                       1510                                                                          GTAACCACCTCACAGATTCCAGCTTCGGAACAAGAGACCCTGGTTAGA398                           ValThrThrSerGlnIleProAlaSerGluGlnGluThrLeuValArg                              152025                                                                        CCAAAGCCATTGCTTTTGAAGTTATTAAAGTCTGTTGGTGCACAAAAA446                           ProLysProLeuLeuLeuLysLeuLeuLysSerValGlyAlaGlnLys                              30354045                                                                      GACACTTATACTATGAAAGAGGTTCTTTTTTATCTTGGCCAGTATATT494                           AspThrTyrThrMetLysGluValLeuPheTyrLeuGlyGlnTyrIle                              505560                                                                        ATGACTAAACGATTATATGATGAGAAGCAACAACATATTGTATATTGT542                           MetThrLysArgLeuTyrAspGluLysGlnGlnHisIleValTyrCys                              657075                                                                        TCAAATGATCTTCTAGGAGATTTGTTTGGCGTGCCAAGCTTCTCTGTG590                           SerAsnAspLeuLeuGlyAspLeuPheGlyValProSerPheSerVal                              808590                                                                        AAAGAGCACAGGAAAATATATACCATGATCTACAGGAACTTGGTAGTA638                           LysGluHisArgLysIleTyrThrMetIleTyrArgAsnLeuValVal                              95100105                                                                      GTCAATCAGCAGGAATCATCGGACTCAGGTACATCTGTGAGTGAGAAC686                           ValAsnGlnGlnGluSerSerAspSerGlyThrSerValSerGluAsn                              110115120125                                                                  AGGTGTCACCTTGAAGGTGGGAGTGATCAAAAGGACCTTGTACAAGAG734                           ArgCysHisLeuGluGlyGlySerAspGlnLysAspLeuValGlnGlu                              130135140                                                                     CTTCAGGAAGAGAAACCTTCATCTTCACATTTGGTTTCTAGACCATCT782                           LeuGlnGluGluLysProSerSerSerHisLeuValSerArgProSer                              145150155                                                                     ACCTCATCTAGAAGGAGAGCAATTAGTGAGACAGAAGAAAATTCAGAT830                           ThrSerSerArgArgArgAlaIleSerGluThrGluGluAsnSerAsp                              160165170                                                                     GAATTATCTGGTGAACGACAAAGAAAACGCCACAAATCTGATAGTATT878                           GluLeuSerGlyGluArgGlnArgLysArgHisLysSerAspSerIle                              175180185                                                                     TCCCTTTCCTTTGATGAAAGCCTGGCTCTGTGTGTAATAAGGGAGATA926                           SerLeuSerPheAspGluSerLeuAlaLeuCysValIleArgGluIle                              190195200205                                                                  TGTTGTGAAAGAAGCAGTAGCAGTGAATCTACAGGGACGCCATCGAAT974                           CysCysGluArgSerSerSerSerGluSerThrGlyThrProSerAsn                              210215220                                                                     CCGGATCTTGATGCTGGTGTAAGTGAACATTCAGGTGATTGGTTGGAT1022                          ProAspLeuAspAlaGlyValSerGluHisSerGlyAspTrpLeuAsp                              225230235                                                                     CAGGATTCAGTTTCAGATCAGTTTAGTGTAGAATTTGAAGTTGAATCT1070                          GlnAspSerValSerAspGlnPheSerValGluPheGluValGluSer                              240245250                                                                     CTCGACTCAGAAGATTATAGCCTTAGTGAAGAAGGACAAGAACTCTCA1118                          LeuAspSerGluAspTyrSerLeuSerGluGluGlyGlnGluLeuSer                              255260265                                                                     GATGAAGATGATGAGGTATATCAAGTTACTGTGTATCAGGCAGGGGAG1166                          AspGluAspAspGluValTyrGlnValThrValTyrGlnAlaGlyGlu                              270275280285                                                                  AGTGATACAGATTCATTTGAAGAAGATCCTGAAATTTCCTTAGCTGAC1214                          SerAspThrAspSerPheGluGluAspProGluIleSerLeuAlaAsp                              290295300                                                                     TATTGGAAATGCACTTCATGCAATGAAATGAATCCCCCCCTTCCATCA1262                          TyrTrpLysCysThrSerCysAsnGluMetAsnProProLeuProSer                              305310315                                                                     CATTGCAACAGATGTTGGGCCCTTCGTGAGAATTGGCTTCCTGAAGAT1310                          HisCysAsnArgCysTrpAlaLeuArgGluAsnTrpLeuProGluAsp                              320325330                                                                     AAAGGGAAAGATAAAGGGGAAATCTCTGAGAAAGCCAAACTGGAAAAC1358                          LysGlyLysAspLysGlyGluIleSerGluLysAlaLysLeuGluAsn                              335340345                                                                     TCAACACAAGCTGAAGAGGGCTTTGATGTTCCTGATTGTAAAAAAACT1406                          SerThrGlnAlaGluGluGlyPheAspValProAspCysLysLysThr                              350355360365                                                                  ATAGTGAATGATTCCAGAGAGTCATGTGTTGAGGAAAATGATGATAAA1454                          IleValAsnAspSerArgGluSerCysValGluGluAsnAspAspLys                              370375380                                                                     ATTACACAAGCTTCACAATCACAAGAAAGTGAAGACTATTCTCAGCCA1502                          IleThrGlnAlaSerGlnSerGlnGluSerGluAspTyrSerGlnPro                              385390395                                                                     TCAACTTCTAGTAGCATTATTTATAGCAGCCAAGAAGATGTGAAAGAG1550                          SerThrSerSerSerIleIleTyrSerSerGlnGluAspValLysGlu                              400405410                                                                     TTTGAAAGGGAAGAAACCCAAGACAAAGAAGAGAGTGTGGAATCTAGT1598                          PheGluArgGluGluThrGlnAspLysGluGluSerValGluSerSer                              415420425                                                                     TTGCCCCTTAATGCCATTGAACCTTGTGTGATTTGTCAAGGTCGACCT1646                          LeuProLeuAsnAlaIleGluProCysValIleCysGlnGlyArgPro                              430435440445                                                                  AAAAATGGTTGCATTGTCCATGGCAAAACAGGACATCTTATGGCCTGC1694                          LysAsnGlyCysIleValHisGlyLysThrGlyHisLeuMetAlaCys                              450455460                                                                     TTTACATGTGCAAAGAAGCTAAAGAAAAGGAATAAGCCCTGCCCAGTA1742                          PheThrCysAlaLysLysLeuLysLysArgAsnLysProCysProVal                              465470475                                                                     TGTAGACAACCAATTCAAATGATTGTGCTAACTTATTTCCCC1784                                CysArgGlnProIleGlnMetIleValLeuThrTyrPhePro                                    480485490                                                                     TAGTTGACCTGTCTATAAGAGAATTATATATTTCTAACTATATAACCCTAGGAATTTAGA1844              CAACCTGAAATTTATTCACATATATCAAAGTGAGAAAATGCCTCAATTCACATAGATTTC1904              TTCTCTTTAGTATAATTGACCTACTTTGGTAGTGGAATAGTGAATACTTACTATAATTTG1964              ACTTGAATATGTAGCTCATCCTTTACACCAACTCCTAATTTTAAATAATTTCTACTCTGT2024              CTTAAATGAGAAGTACTTGGTTTTTTTTTTCTTAAATATGTATATGACATTTAAATGTAA2084              CTTATTATTTTTTTTGAGACCGAGTCTTGCTCTGTTACCCAGGCTGGAGTGCAGTGGGTG2144              ATCTTGGCTCACTGCAAGCTCTGCCCTCCCCGGGTTCGCACCATTCTCCTGCCTCAGCCT2204              CCCAATTAGCTTGGCCTACAGTCATCTGCCACCACACCTGGCTAATTTTTTGTACTTTTA2264              GTAGAGACAGGGTTTCACCGTGTTAGCCAGGATGGTCTCGATCTCCTGACCTCGTGATCC2324              GCCCACCTCGGCCTCCCAAAGTGCTGGGATTACAGGCATGAGCCACCG2372                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 491 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       MetCysAsnThrAsnMetSerValProThrAspGlyAlaValThrThr                              151015                                                                        SerGlnIleProAlaSerGluGlnGluThrLeuValArgProLysPro                              202530                                                                        LeuLeuLeuLysLeuLeuLysSerValGlyAlaGlnLysAspThrTyr                              354045                                                                        ThrMetLysGluValLeuPheTyrLeuGlyGlnTyrIleMetThrLys                              505560                                                                        ArgLeuTyrAspGluLysGlnGlnHisIleValTyrCysSerAsnAsp                              65707580                                                                      LeuLeuGlyAspLeuPheGlyValProSerPheSerValLysGluHis                              859095                                                                        ArgLysIleTyrThrMetIleTyrArgAsnLeuValValValAsnGln                              100105110                                                                     GlnGluSerSerAspSerGlyThrSerValSerGluAsnArgCysHis                              115120125                                                                     LeuGluGlyGlySerAspGlnLysAspLeuValGlnGluLeuGlnGlu                              130135140                                                                     GluLysProSerSerSerHisLeuValSerArgProSerThrSerSer                              145150155160                                                                  ArgArgArgAlaIleSerGluThrGluGluAsnSerAspGluLeuSer                              165170175                                                                     GlyGluArgGlnArgLysArgHisLysSerAspSerIleSerLeuSer                              180185190                                                                     PheAspGluSerLeuAlaLeuCysValIleArgGluIleCysCysGlu                              195200205                                                                     ArgSerSerSerSerGluSerThrGlyThrProSerAsnProAspLeu                              210215220                                                                     AspAlaGlyValSerGluHisSerGlyAspTrpLeuAspGlnAspSer                              225230235240                                                                  ValSerAspGlnPheSerValGluPheGluValGluSerLeuAspSer                              245250255                                                                     GluAspTyrSerLeuSerGluGluGlyGlnGluLeuSerAspGluAsp                              260265270                                                                     AspGluValTyrGlnValThrValTyrGlnAlaGlyGluSerAspThr                              275280285                                                                     AspSerPheGluGluAspProGluIleSerLeuAlaAspTyrTrpLys                              290295300                                                                     CysThrSerCysAsnGluMetAsnProProLeuProSerHisCysAsn                              305310315320                                                                  ArgCysTrpAlaLeuArgGluAsnTrpLeuProGluAspLysGlyLys                              325330335                                                                     AspLysGlyGluIleSerGluLysAlaLysLeuGluAsnSerThrGln                              340345350                                                                     AlaGluGluGlyPheAspValProAspCysLysLysThrIleValAsn                              355360365                                                                     AspSerArgGluSerCysValGluGluAsnAspAspLysIleThrGln                              370375380                                                                     AlaSerGlnSerGlnGluSerGluAspTyrSerGlnProSerThrSer                              385390395400                                                                  SerSerIleIleTyrSerSerGlnGluAspValLysGluPheGluArg                              405410415                                                                     GluGluThrGlnAspLysGluGluSerValGluSerSerLeuProLeu                              420425430                                                                     AsnAlaIleGluProCysValIleCysGlnGlyArgProLysAsnGly                              435440445                                                                     CysIleValHisGlyLysThrGlyHisLeuMetAlaCysPheThrCys                              450455460                                                                     AlaLysLysLeuLysLysArgAsnLysProCysProValCysArgGln                              465470475480                                                                  ProIleGlnMetIleValLeuThrTyrPhePro                                             485490                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1710 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mus musculus                                                    (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 202..1668                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GAGGAGCCGCCGCCTTCTCGTCGCTCGAGCTCTGGACGACCATGGTCGCTCAGGCCCCGT60                CCGCGGGGCCTCCGCGCTCCCCGTGAAGGGTCGGAAGATGCGCGGGAAGTAGCAGCCGTC120               TGCTGGGCGAGCGGGAGACCGACCGGACACCCCTGGGGGACCCTCTCGGATCACCGCGCT180               TCTCCTGCGGCCTCCAGGCCAATGTGCAATACCAACATGTCTGTGTCTACC231                        MetCysAsnThrAsnMetSerValSerThr                                                1510                                                                          GAGGGTGCTGCAAGCACCTCACAGATTCCAGCTTCGGAACAAGAGACT279                           GluGlyAlaAlaSerThrSerGlnIleProAlaSerGluGlnGluThr                              152025                                                                        CTGGTTAGACCAAAACCATTGCTTTTGAAGTTGTTAAAGTCCGTTGGA327                           LeuValArgProLysProLeuLeuLeuLysLeuLeuLysSerValGly                              303540                                                                        GCGCAAAACGACACTTACACTATGAAAGAGATTATATTTTATATTGGC375                           AlaGlnAsnAspThrTyrThrMetLysGluIleIlePheTyrIleGly                              455055                                                                        CAGTATATTATGACTAAGAGGTTATATGACGAGAAGCAGCAGCACATT423                           GlnTyrIleMetThrLysArgLeuTyrAspGluLysGlnGlnHisIle                              606570                                                                        GTGTATTGTTCAAATGATCTCCTAGGAGATGTGTTTGGAGTCCCGAGT471                           ValTyrCysSerAsnAspLeuLeuGlyAspValPheGlyValProSer                              75808590                                                                      TTCTCTGTGAAGGAGCACAGGAAAATATATGCAATGATCTACAGAAAT519                           PheSerValLysGluHisArgLysIleTyrAlaMetIleTyrArgAsn                              95100105                                                                      TTAGTGGCTGTAAGTCAGCAAGACTCTGGCACATCGCTGAGTGAGAGC567                           LeuValAlaValSerGlnGlnAspSerGlyThrSerLeuSerGluSer                              110115120                                                                     AGACGTCAGCCTGAAGGTGGGAGTGATCTGAAGGATCCTTTGCAAGCG615                           ArgArgGlnProGluGlyGlySerAspLeuLysAspProLeuGlnAla                              125130135                                                                     CCACCAGAAGAGAAACCTTCATCTTCTGATTTAATTTCTAGACTGTCT663                           ProProGluGluLysProSerSerSerAspLeuIleSerArgLeuSer                              140145150                                                                     ACCTCATCTAGAAGGAGATCCATTAGTGAGACAGAAGAGAACACAGAT711                           ThrSerSerArgArgArgSerIleSerGluThrGluGluAsnThrAsp                              155160165170                                                                  GAGCTACCTGGGGAGCGGCACCGGAAGCGCCGCAGGTCCCTGTCCTTT759                           GluLeuProGlyGluArgHisArgLysArgArgArgSerLeuSerPhe                              175180185                                                                     GATCCGAGCCTGGGTCTGTGTGAGCTGAGGGAGATGTGCAGCGGCGGC807                           AspProSerLeuGlyLeuCysGluLeuArgGluMetCysSerGlyGly                              190195200                                                                     ACGAGCAGCAGTAGCAGCAGCAGCAGCGAGTCCACAGAGACGCCCTCG855                           ThrSerSerSerSerSerSerSerSerGluSerThrGluThrProSer                              205210215                                                                     CATCAGGATCTTGACGATGGCGTAAGTGAGCATTCTGGTGATTGCCTG903                           HisGlnAspLeuAspAspGlyValSerGluHisSerGlyAspCysLeu                              220225230                                                                     GATCAGGATTCAGTTTCTGATCAGTTTAGCGTGGAATTTGAAGTTGAG951                           AspGlnAspSerValSerAspGlnPheSerValGluPheGluValGlu                              235240245250                                                                  TCTCTGGACTCGGAAGATTACAGCCTGAGTGACGAAGGGCACGAGCTC999                           SerLeuAspSerGluAspTyrSerLeuSerAspGluGlyHisGluLeu                              255260265                                                                     TCAGATGAGGATGATGAGGTCTATCGGGTCACAGTCTATCAGACAGGA1047                          SerAspGluAspAspGluValTyrArgValThrValTyrGlnThrGly                              270275280                                                                     GAAAGCGATACAGACTCTTTTGAAGGAGATCCTGAGATTTCCTTAGCT1095                          GluSerAspThrAspSerPheGluGlyAspProGluIleSerLeuAla                              285290295                                                                     GACTATTGGAAGTGTACCTCATGCAATGAAATGAATCCTCCCCTTCCA1143                          AspTyrTrpLysCysThrSerCysAsnGluMetAsnProProLeuPro                              300305310                                                                     TCACACTGCAAAAGATGCTGGACCCTTCGTGAGAACTGGCTTCCAGAC1191                          SerHisCysLysArgCysTrpThrLeuArgGluAsnTrpLeuProAsp                              315320325330                                                                  GATAAGGGGAAAGATAAAGTGGAAATCTCTGAAAAAGCCAAACTGGAA1239                          AspLysGlyLysAspLysValGluIleSerGluLysAlaLysLeuGlu                              335340345                                                                     AACTCAGCTCAGGCAGAAGAAGGCTTGGATGTGCCTGATGGCAAAAAG1287                          AsnSerAlaGlnAlaGluGluGlyLeuAspValProAspGlyLysLys                              350355360                                                                     CTGACAGAGAATGATGCTAAAGAGCCATGTGCTGAGGAGGACAGCGAG1335                          LeuThrGluAsnAspAlaLysGluProCysAlaGluGluAspSerGlu                              365370375                                                                     GAGAAGGCCGAACAGACGCCCCTGTCCCAGGAGAGTGACGACTATTCC1383                          GluLysAlaGluGlnThrProLeuSerGlnGluSerAspAspTyrSer                              380385390                                                                     CAACCATCGACTTCCAGCAGCATTGTTTATAGCAGCCAAGAAAGCGTG1431                          GlnProSerThrSerSerSerIleValTyrSerSerGlnGluSerVal                              395400405410                                                                  AAAGAGTTGAAGGAGGAAACGCAGCACAAAGACGAGAGTGTGGAATCT1479                          LysGluLeuLysGluGluThrGlnHisLysAspGluSerValGluSer                              415420425                                                                     AGCTTCTCCCTGAATGCCATCGAACCATGTGTGATCTGCCAGGGGCGG1527                          SerPheSerLeuAsnAlaIleGluProCysValIleCysGlnGlyArg                              430435440                                                                     CCTAAAAATGGCTGCATTGTTCACGGCAAGACTGGACACCTCATGTCA1575                          ProLysAsnGlyCysIleValHisGlyLysThrGlyHisLeuMetSer                              445450455                                                                     TGTTTCACGTGTGCAAAGAAGCTAAAAAAAAGAAACAAGCCCTGCCCA1623                          CysPheThrCysAlaLysLysLeuLysLysArgAsnLysProCysPro                              460465470                                                                     GTGTGCAGACAGCCAATCCAAATGATTGTGCTAAGTTACTTCAAC1668                             ValCysArgGlnProIleGlnMetIleValLeuSerTyrPheAsn                                 475480485                                                                     TAGCTGACCTGCTCACAAAAATAGAATTTTATATTTCTAACT1710                                (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 489 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       MetCysAsnThrAsnMetSerValSerThrGluGlyAlaAlaSerThr                              151015                                                                        SerGlnIleProAlaSerGluGlnGluThrLeuValArgProLysPro                              202530                                                                        LeuLeuLeuLysLeuLeuLysSerValGlyAlaGlnAsnAspThrTyr                              354045                                                                        ThrMetLysGluIleIlePheTyrIleGlyGlnTyrIleMetThrLys                              505560                                                                        ArgLeuTyrAspGluLysGlnGlnHisIleValTyrCysSerAsnAsp                              65707580                                                                      LeuLeuGlyAspValPheGlyValProSerPheSerValLysGluHis                              859095                                                                        ArgLysIleTyrAlaMetIleTyrArgAsnLeuValAlaValSerGln                              100105110                                                                     GlnAspSerGlyThrSerLeuSerGluSerArgArgGlnProGluGly                              115120125                                                                     GlySerAspLeuLysAspProLeuGlnAlaProProGluGluLysPro                              130135140                                                                     SerSerSerAspLeuIleSerArgLeuSerThrSerSerArgArgArg                              145150155160                                                                  SerIleSerGluThrGluGluAsnThrAspGluLeuProGlyGluArg                              165170175                                                                     HisArgLysArgArgArgSerLeuSerPheAspProSerLeuGlyLeu                              180185190                                                                     CysGluLeuArgGluMetCysSerGlyGlyThrSerSerSerSerSer                              195200205                                                                     SerSerSerGluSerThrGluThrProSerHisGlnAspLeuAspAsp                              210215220                                                                     GlyValSerGluHisSerGlyAspCysLeuAspGlnAspSerValSer                              225230235240                                                                  AspGlnPheSerValGluPheGluValGluSerLeuAspSerGluAsp                              245250255                                                                     TyrSerLeuSerAspGluGlyHisGluLeuSerAspGluAspAspGlu                              260265270                                                                     ValTyrArgValThrValTyrGlnThrGlyGluSerAspThrAspSer                              275280285                                                                     PheGluGlyAspProGluIleSerLeuAlaAspTyrTrpLysCysThr                              290295300                                                                     SerCysAsnGluMetAsnProProLeuProSerHisCysLysArgCys                              305310315320                                                                  TrpThrLeuArgGluAsnTrpLeuProAspAspLysGlyLysAspLys                              325330335                                                                     ValGluIleSerGluLysAlaLysLeuGluAsnSerAlaGlnAlaGlu                              340345350                                                                     GluGlyLeuAspValProAspGlyLysLysLeuThrGluAsnAspAla                              355360365                                                                     LysGluProCysAlaGluGluAspSerGluGluLysAlaGluGlnThr                              370375380                                                                     ProLeuSerGlnGluSerAspAspTyrSerGlnProSerThrSerSer                              385390395400                                                                  SerIleValTyrSerSerGlnGluSerValLysGluLeuLysGluGlu                              405410415                                                                     ThrGlnHisLysAspGluSerValGluSerSerPheSerLeuAsnAla                              420425430                                                                     IleGluProCysValIleCysGlnGlyArgProLysAsnGlyCysIle                              435440445                                                                     ValHisGlyLysThrGlyHisLeuMetSerCysPheThrCysAlaLys                              450455460                                                                     LysLeuLysLysArgAsnLysProCysProValCysArgGlnProIle                              465470475480                                                                  GlnMetIleValLeuSerTyrPheAsn                                                   485                                                                           (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 393 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: YES                                                       (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS: Buchman, et al.,                                                 (C) JOURNAL: Gene                                                             (D) VOLUME: 70                                                                (F) PAGES: 245-252                                                            (G) DATE: 1988                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       MetGluGluProGlnSerAspProSerValGluProProLeuSerGln                              151015                                                                        GluThrPheSerAspLeuTrpLysLeuLeuProGluAsnAsnValLeu                              202530                                                                        SerProLeuProSerGlnAlaMetAspAspLeuMetLeuSerProAsp                              354045                                                                        AspIleGluGlnTrpPheThrGluAspProGlyProAspGluAlaPro                              505560                                                                        ArgMetProGluAlaAlaProProValAlaProAlaProAlaAlaPro                              65707580                                                                      ThrProAlaAlaProAlaProAlaProSerTrpProLeuSerSerSer                              859095                                                                        ValProSerGlnLysThrTyrGlnGlySerTyrGlyPheArgLeuGly                              100105110                                                                     PheLeuHisSerGlyThrAlaLysSerValThrCysThrTyrSerPro                              115120125                                                                     AlaLeuAsnLysMetPheCysGlnLeuAlaLysThrCysProValGln                              130135140                                                                     LeuTrpValAspSerThrProProProGlyThrArgValArgAlaMet                              145150155160                                                                  AlaIleTyrLysGlnSerGlnHisMetThrGluValValArgArgCys                              165170175                                                                     ProHisHisGluArgCysSerAspSerAspGlyLeuAlaProProGln                              180185190                                                                     HisLeuIleArgValGluGlyAsnLeuArgValGluTyrLeuAspAsp                              195200205                                                                     ArgAsnThrPheArgHisSerValValValProTyrGluProProGlu                              210215220                                                                     ValGlySerAspCysThrThrIleHisTyrAsnTyrMetCysAsnSer                              225230235240                                                                  SerCysMetGlyGlyMetAsnArgArgProIleLeuThrIleIleThr                              245250255                                                                     LeuGluAspSerSerGlyAsnLeuLeuGlyArgAsnSerPheGluVal                              260265270                                                                     ArgValCysAlaCysProGlyArgAspArgArgThrGluGluGluAsn                              275280285                                                                     LeuArgLysLysGlyGluProHisHisGluLeuProProGlySerThr                              290295300                                                                     LysArgAlaLeuProAsnAsnThrSerSerSerProGlnProLysLys                              305310315320                                                                  LysProLeuAspGlyGluTyrPheThrLeuGlnIleArgGlyArgGlu                              325330335                                                                     ArgPheGluMetPheArgGluLeuAsnGluAlaLeuGluLeuLysAsp                              340345350                                                                     AlaGlnAlaGlyLysGluProGlyGlySerArgAlaHisSerSerHis                              355360365                                                                     LeuLysSerLysLysGlyGlnSerThrSerArgHisLysLysLeuMet                              370375380                                                                     PheLysThrGluGlyProAspSerAsp                                                   385390                                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 393 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: YES                                                       (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS: Harris, et al.,                                                  (C) JOURNAL: Mol. Cell. Biol.                                                 (D) VOLUME: 6                                                                 (E) ISSUE: 12                                                                 (F) PAGES: 4650-4656                                                          (G) DATE: 1986                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       MetGluGluProGlnSerAspProSerValGluProProLeuSerGln                              151015                                                                        GluThrPheSerAspLeuTrpLysLeuLeuProGluAsnAsnValLeu                              202530                                                                        SerProLeuProSerGlnAlaMetAspAspLeuMetLeuSerProAsp                              354045                                                                        AspIleGluGlnTrpPheThrGluAspProGlyProAspGluAlaPro                              505560                                                                        ArgMetProGluAlaAlaProArgValAlaProAlaProAlaThrPro                              65707580                                                                      ThrProAlaAlaProAlaProAlaProSerTrpProLeuSerSerSer                              859095                                                                        ValProSerGlnLysThrTyrGlnGlySerTyrGlyPheArgLeuGly                              100105110                                                                     PheLeuHisSerGlyThrAlaLysSerValThrCysThrTyrSerPro                              115120125                                                                     AlaLeuAsnLysMetPheCysGlnLeuAlaLysThrCysProValGln                              130135140                                                                     LeuTrpValAspSerThrProProProGlyThrArgValArgAlaMet                              145150155160                                                                  AlaIleTyrLysGlnSerGlnHisMetThrGluValValArgArgCys                              165170175                                                                     ProHisHisGluArgCysSerAspSerAspGlyLeuAlaProProGln                              180185190                                                                     HisLeuIleArgValGluGlyAsnLeuArgValGluTyrLeuAspAsp                              195200205                                                                     ArgAsnThrPheArgHisSerValValValProTyrGluProProGlu                              210215220                                                                     ValGlySerAspCysThrThrIleHisTyrAsnTyrMetCysAsnSer                              225230235240                                                                  SerCysMetGlyGlyMetAsnArgArgProIleLeuThrIleIleThr                              245250255                                                                     LeuGluAspSerSerGlyAsnLeuLeuGlyArgAsnSerPheGluVal                              260265270                                                                     ArgValCysAlaCysProGlyArgAspArgArgThrGluGluGluAsn                              275280285                                                                     LeuArgLysLysGlyGluProHisHisGluLeuProProGlySerThr                              290295300                                                                     LysArgAlaLeuProAsnAsnThrSerSerSerProGlnProLysLys                              305310315320                                                                  LysProLeuAspGlyGluTyrPheThrLeuGlnIleArgGlyArgGlu                              325330335                                                                     ArgPheGluMetPheArgGluLeuAsnGluAlaLeuGluLeuLysAsp                              340345350                                                                     AlaGlnAlaGlyLysGluProGlyGlySerArgAlaHisSerSerHis                              355360365                                                                     LeuLysSerLysLysGlyGlnSerThrSerArgHisLysLysLeuMet                              370375380                                                                     PheLysThrGluGlyProAspSerAsp                                                   385390                                                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 393 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: YES                                                       (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS: Harris, et al.,                                                  (C) JOURNAL: Mol. Cell. Biol.                                                 (D) VOLUME: 6                                                                 (E) ISSUE: 12                                                                 (F) PAGES: 4650-4656                                                          (G) DATE: 1986                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       MetGluGluProGlnSerAspProSerValGluProProLeuSerGln                              151015                                                                        GluThrPheSerAspLeuTrpLysLeuLeuProGluAsnAsnValLeu                              202530                                                                        SerProLeuProSerGlnAlaMetAspAspLeuMetLeuSerProAsp                              354045                                                                        AspIleGluGlnTrpPheThrGluAspProGlyProAspGluAlaPro                              505560                                                                        ArgMetProGluAlaAlaProProValAlaProAlaProAlaThrPro                              65707580                                                                      ThrProAlaAlaProAlaProAlaProSerTrpProLeuSerSerSer                              859095                                                                        ValProSerGlnLysThrTyrGlnGlySerTyrGlyPheArgLeuGly                              100105110                                                                     PheLeuHisSerGlyThrAlaLysSerValThrCysThrTyrSerPro                              115120125                                                                     AlaLeuAsnLysMetPheCysGlnLeuAlaLysThrCysProValGln                              130135140                                                                     LeuTrpValAspSerThrProProProGlyThrArgValArgAlaMet                              145150155160                                                                  AlaIleTyrLysGlnSerGlnHisMetThrGluValValArgArgCys                              165170175                                                                     ProHisHisGluArgCysSerAspSerAspGlyLeuAlaProProGln                              180185190                                                                     HisLeuIleArgValGluGlyAsnLeuArgValGluTyrLeuAspAsp                              195200205                                                                     ArgAsnThrPheArgHisSerValValValProTyrGluProProGlu                              210215220                                                                     ValGlySerAspCysThrThrIleHisTyrAsnTyrMetCysAsnSer                              225230235240                                                                  SerCysMetGlyGlyMetAsnArgArgProIleLeuThrIleIleThr                              245250255                                                                     LeuGluAspSerSerGlyAsnLeuLeuGlyArgAsnSerPheGluVal                              260265270                                                                     ArgValCysAlaCysProGlyArgAspArgArgThrGluGluGluAsn                              275280285                                                                     LeuArgLysLysGlyGluProHisHisGluLeuProProGlySerThr                              290295300                                                                     LysArgAlaLeuProAsnAsnThrSerSerSerProGlnProLysLys                              305310315320                                                                  LysProLeuAspGlyGluTyrPheThrLeuGlnIleArgGlyArgGlu                              325330335                                                                     ArgPheGluMetPheArgGluLeuAsnGluAlaLeuGluLeuLysAsp                              340345350                                                                     AlaGlnAlaGlyLysGluProGlyGlySerArgAlaHisSerSerHis                              355360365                                                                     LeuLysSerLysLysGlyGlnSerThrSerArgHisLysLysLeuMet                              370375380                                                                     PheLysThrGluGlyProAspSerAsp                                                   385390                                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 393 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: YES                                                       (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS: Lamb, P.                                                         Crawford, L.                                                                  (C) JOURNAL: Mol. Cell. Biol.                                                 (D) VOLUME: 6                                                                 (E) ISSUE: 5                                                                  (F) PAGES: 1379-1385                                                          (G) DATE: 1986                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       MetGluGluProGlnSerAspProSerValGluProProLeuSerGln                              151015                                                                        GluThrPheSerAspLeuTrpLysLeuLeuProGluAsnAsnValLeu                              202530                                                                        SerProLeuProSerGlnAlaMetAspAspLeuMetLeuSerProAsp                              354045                                                                        AspIleGluGlnTrpPheThrGluAspProGlyProAspGluAlaPro                              505560                                                                        ArgMetProGluAlaAlaProArgValAlaProGlyProAlaAlaPro                              65707580                                                                      ThrProAlaAlaProAlaProAlaProSerTrpProLeuSerSerSer                              859095                                                                        ValProSerGlnLysThrTyrGlnGlySerTyrGlyPheArgLeuGly                              100105110                                                                     PheLeuHisSerGlyThrAlaLysSerValThrCysThrTyrSerPro                              115120125                                                                     AlaLeuAsnLysMetPheCysGlnLeuAlaLysThrCysProValGln                              130135140                                                                     LeuTrpValAspSerThrProProProGlyThrArgValArgAlaMet                              145150155160                                                                  AlaIleTyrLysGlnSerGlnHisMetThrGluValValArgArgCys                              165170175                                                                     ProHisHisGluArgCysSerAspSerAspGlyLeuAlaProProGln                              180185190                                                                     HisLeuIleArgValGluGlyAsnLeuArgValGluTyrLeuAspAsp                              195200205                                                                     ArgAsnThrPheArgHisSerValValValProTyrGluProProGlu                              210215220                                                                     ValGlySerAspCysThrThrIleHisTyrAsnTyrMetCysAsnSer                              225230235240                                                                  SerCysMetGlyGlyMetAsnArgArgProIleLeuThrIleIleThr                              245250255                                                                     LeuGluAspSerSerGlyAsnLeuLeuGlyArgAsnSerPheGluVal                              260265270                                                                     ArgValCysAlaCysProGlyArgAspArgArgThrGluGluGluAsn                              275280285                                                                     LeuArgLysLysGlyGluProHisHisGluLeuProProGlySerThr                              290295300                                                                     LysArgAlaLeuProAsnAsnThrSerSerSerProGlnProLysLys                              305310315320                                                                  LysProLeuAspGlyGluTyrPheThrLeuGlnIleArgGlyArgGlu                              325330335                                                                     ArgPheGluMetPheArgGluLeuAsnGluAlaLeuGluLeuLysAsp                              340345350                                                                     AlaGlnAlaGlyLysGluProGlyGlySerArgAlaHisSerSerHis                              355360365                                                                     LeuLysSerLysLysGlyGlnSerThrSerArgHisLysLysLeuMet                              370375380                                                                     PheLysThrGluGlyProAspSerAsp                                                   385390                                                                        __________________________________________________________________________

We claim:
 1. A method for inhibiting the growth of tumor cells whichcontain a human MDM2 gene amplification, comprising:administering totumor cells which contain a human MDM2 gene amplification a DNA moleculewhich expresses a polypeptide consisting of a portion of p53, whereinsaid polypeptide comprises amino acids 1-50 of p53 as shown in SEQ IDNO:1, said polypeptide being capable of binding to human MDM2 as shownin SEQ ID NO:3.
 2. A method for inhibiting the growth of tumor cellswhich contain a human MDM2 gene amplification, comprising:administeringto tumor cells which contain a human MDM2 gene amplification a DNAmolecule which expresses a polypeptide consisting of a portion of p53,said portion comprising amino acids 13-41 of p53 as shown in SEQ ID NO:1and at least nine additional p53 residues on either the amino or carboxyterminal side, wherein said polypeptide lacks the homo-oligomerizationdomain of p53, and said polypeptide is capable of binding to human MDM2as shown in SEQ ID NO:3.
 3. A method for inhibiting the growth of tumorcells which contain a human MDM2 gene amplification,comprising:administering to tumor cells which contain a human MDM2 geneamplification a DNA molecule which expresses a polypeptide consisting ofa portion of p53, said portion comprising amino acids 13-41 of p53 asshown in SEQ ID NO:1 and at least nine additional p53 residues on eitherthe amino or carboxy terminal side, said polypeptide being capable ofbinding to human MDM2 as shown in SEQ ID NO:3, wherein said polypeptidelacks amino acids 138-393 of p53as shown in SEQ ID NO:6, 7, 8, or 9 andsaid polypeptide is capable of binding to human MDM2.